HIV-related morbidity/mortality as well as disease progress of AIDS, were successfully controlled by clinical use of HIV protease inhibitors. However, current HIV protease inhibitors, from the first generation drug indinavir to the most recent lopinavir and atazanavir, are all peptidomimetic and transition state analogs in nature. They share similar chemical structures and occupy very narrow chemical space. Therefore, they share overlapping drug-resistance profiles. In order to more effectively combat drug resistance in AIDS, there is an urgent need of next-generation HIV protease inhibitors that would possess entirely different chemical structures. Recently, in our laboratory we have developed a prototype of E. coli based HIV protease assay that we will continue to develop into a HTS platform, and will use it as an initial screen to identify hit compounds from diverse chemical libraries for the discovery of new HIV protease inhibitors. In this proposal, we plan to develop a human T cell based assay. We will use inducible retroviral vectors to transfer DNA sequences encoding the HIV protease sequence and the same reporter that was used in the E. coli assay, i.e. beta-galactosidase gene into human CD4+ T cells to establish stable cell lines. Based on statistical analysis of experimental data, we will choose those most robust and reproducible assay conditions to establish protocols that can be adapted for automated molecular screening in a high throughput manner. Also, in the project we plan to carry out a pilot study with the human CD4+ T cell based assay. That involves screening a small compound library, namely Spectrum Collection, of 2,000 compounds. To confirm active compounds identified in such a cell-based assay, we will test these compounds (hits) in the in vitro HIV protease inhibition assay. Finally, we will perform in vitro inhibition assays for these HIV protease inhibitors against a panel of human aspartic enzymes to evaluate their specificity profiles. We anticipate that after this human CD4+ T cell based HIV protease assay is fully developed and validated it can be most useful in automated screening of chemical analogs of original hits during the lead optimization stage in order to attain HIV protease inhibitors with better ADME properties. Active compounds meeting the criteria for potency and specificity can be selected and prioritized for future drug development into useful therapeutic agents for treating multidrug-resistant AIDS community through mechanisms discussed in the NIH Roadmap Initiative.